*Target (protein/gene name): Trypanothione Reductase *NCBI Gene # or RefSeq#: 22664 *Protein ID (NP or XP #) or Wolbachia#: LmjF05.0350 *Organism (including strain): Leishmania major Etiologic Risk Group (see link below): Risk Group 2 - Parasitic Agent *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Leishmania major is a protozoan parasite that causes Leishmaniasis which is characterized by a cutaneous ulcer that can last from a few weeks to a few months. Leishmania major is mostly found in the Eastern Hemisphere because the protozoan is carried by infected sandflies. Leishmania major utilizes antioxidant enzymes and lysozyme inhibitors to successfully thrive in the body of sandflies and then the protozoan is than transferred into humans through sandfly bites. There are currently vaccines being developed for this disease but they have not gone global due to lack of knowledge of specific antigens that are involved in Lesihmaniasis. Most people who have Leishmaniasis will heal and development an immunity to any future infections. Essentiality of this protein:
Gene/Ortholog: mtu2902 (OG4_10249); Phenotype: essential; Source study: nmpdr Gene/Ortholog: eco3379 (OG4_10249); Phenotype: essential; Source study: gerdes Complex of proteins?: No, works alone. Druggable Target: Yes *EC#: 1. 8. 1. 12 Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.8.1.12
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Colorimetric plate assay. (The assay can be performed in 1 mL volume and measured using a spectrophotometer or in 100 ìl volume in a 96 well plate using an ELISA reader.
Caspase 3 Colorimetric Assay Kit provides the reagents needed for a quick and efficient detection of caspase 3 activity in cell lysates and in purified preparations of caspase 3.
The Caspase 3 Colorimetric Assay Kit is based on the hydrolysis of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA) moiety. p-Nitroaniline is detected at 405 nm (εmM=10.5). The concentration of the pNA released from the substrate is calculated from either the absorbance values at 405 nm or from a calibration curve prepared with pNA standards (pNA standard included with the kit).) (Quoted from SigmaAldrich)
-- link to Sigma (or other company) page for assay or assay reagents (substrates) http://www.sigmaaldrich.com/catalog/product/sigma/casp3c?lang=en®ion=US -- link (or citation) to paper that contains assay information http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155241/ -- List cost and quantity of substrate reagents and supplier The Caspase 3 Colorimetric Assay Kit gives the substrate reagents of caspase 3 and one hundred 96 multi welled colorimetric plate assay. Allows for 1000 multiwelled tests or 100 regular tests. Costs 612 USD Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 2JK6 -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: 491 ---- Max % Identities: 470/491 96% ---- % Positives: 481/491 97% ---- Chain used for homology:
MSKIFDLVVIGAGSGF
VVNVRESADPA
VRESADP Current Inhibitors: FLAVIN-ADENINE DINUCLEOTIDE
http://www.sciencedirect.com/science/article/pii/S1046592804004188 Expression Information (has it been expressed in bacterial cells): Yes expressed through BL21(DE3) Purification Method: The gene was expressed in E. Coli then the induced culture was centrifuged at 6000rpm for 20 seconds. The cell pellet was suspended in a buffer and sonicated at a power of 50W. Then centrifuged again and the supernatant was put through a column while utilzing PBS buffer and thrombin. These were used to create elutions of the proteins. Image of protein (PyMol with features delineated and shown separately):
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MMSKIFDLVVIGAGSGGLEAAWNAATLYKKRVAVIDVQMVHGPPFFSALGGTCVNVGCVP
*length of your protein in Amino Acids: 491 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 53867.8 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 21860/ (M cm) TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. Gene ID: 5648897
*GC% Content for gene (codon optimized): 51.08% Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
"Leishmania Major » Protozoan and Helminth Parasites » Pathogen Profile Dictionary."Leishmania Major » Protozoan and Helminth Parasites » Pathogen Profile Dictionary. . <http://www.ppdictionary.com/parasites/major.htm>. (Accessed 4/25/2013)
*NCBI Gene # or RefSeq#: 22664
*Protein ID (NP or XP #) or Wolbachia#: LmjF05.0350
*Organism (including strain): Leishmania major
Etiologic Risk Group (see link below): Risk Group 2 - Parasitic Agent
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmania major is a protozoan parasite that causes Leishmaniasis which is characterized by a cutaneous ulcer that can last from a few weeks to a few months. Leishmania major is mostly found in the Eastern Hemisphere because the protozoan is carried by infected sandflies. Leishmania major utilizes antioxidant enzymes and lysozyme inhibitors to successfully thrive in the body of sandflies and then the protozoan is than transferred into humans through sandfly bites. There are currently vaccines being developed for this disease but they have not gone global due to lack of knowledge of specific antigens that are involved in Lesihmaniasis. Most people who have Leishmaniasis will heal and development an immunity to any future infections.
Essentiality of this protein:
Gene/Ortholog: mtu2902 (OG4_10249); Phenotype: essential; Source study: nmpdr
Gene/Ortholog: eco3379 (OG4_10249); Phenotype: essential; Source study: gerdes
Complex of proteins?: No, works alone.
Druggable Target: Yes
*EC#: 1. 8. 1. 12
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.8.1.12
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Colorimetric plate assay.
(The assay can be performed in 1 mL volume and measured using a spectrophotometer or in 100 ìl volume in a 96 well plate using an ELISA reader.
Caspase 3 Colorimetric Assay Kit provides the reagents needed for a quick and efficient detection of caspase 3 activity in cell lysates and in purified preparations of caspase 3.
The Caspase 3 Colorimetric Assay Kit is based on the hydrolysis of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA) moiety. p-Nitroaniline is detected at 405 nm (εmM=10.5). The concentration of the pNA released from the substrate is calculated from either the absorbance values at 405 nm or from a calibration curve prepared with pNA standards (pNA standard included with the kit).) (Quoted from SigmaAldrich)
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/product/sigma/casp3c?lang=en®ion=US
-- link (or citation) to paper that contains assay information
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155241/
-- List cost and quantity of substrate reagents and supplier
The Caspase 3 Colorimetric Assay Kit gives the substrate reagents of caspase 3 and one hundred 96 multi welled colorimetric plate assay. Allows for 1000 multiwelled tests or 100 regular tests.
Costs 612 USD
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2JK6
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 491
---- Max % Identities: 470/491 96%
---- % Positives: 481/491 97%
---- Chain used for homology:
MSKIFDLVVIGAGSGF
VVNVRESADPA
VRESADP
Current Inhibitors: FLAVIN-ADENINE DINUCLEOTIDE
http://www.sciencedirect.com/science/article/pii/S1046592804004188
Expression Information (has it been expressed in bacterial cells): Yes expressed through BL21(DE3)
Purification Method: The gene was expressed in E. Coli then the induced culture was centrifuged at 6000rpm for 20 seconds. The cell pellet was suspended in a buffer and sonicated at a power of 50W. Then centrifuged again and the supernatant was put through a column while utilzing PBS buffer and thrombin. These were used to create elutions of the proteins.
Image of protein (PyMol with features delineated and shown separately):
MMSKIFDLVVIGAGSGGLEAAWNAATLYKKRVAVIDVQMVHGPPFFSALGGTCVNVGCVP
KKLMVTGAQYMEHLRESAGFGWEFDRTTLRAEWKKLIAVKDEAVLNINKSYEEMFRDTEG
LEFFLGWGSLESKNVVNVRESADPASAVKERLETENILLASGSWPHMPNIPGIEHCISSN
EAFYLPEPPRRVLTVGGGFISVEFAGIFNAYKPKDGQVTLCYRGEMILRGFDHTLREELT
KQLTANGIQILTKENPAKVELNADGSKSVTFESGKKMDFDLVMMAIGRSPRTKDLQLQNA
GVMIKNGGVQVDEYSRTNVSNIYAIGDVTNRVMLTPVAINEAAALVDTVFGTNPRKTDHT
RVASAVFSIPPIGTCGLIEEVASKRYEVVAVYLSSFTPLMHNISGSKYKTFVAKIITNHS
DGTVLGVHLLGDNAPEIIQGVGICLKLNAKISDFYNTIGVHPTSAEELCSMRTPSYYYVK
GEKMEKPSEASL
*length of your protein in Amino Acids: 491 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 53867.8 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 21860/ (M cm)
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Gene ID: 5648897
*CDS Gene Sequence (paste as text only):
1 atgtcccgcg cgtacgacct cgtggtgctt ggcgccggat ctggaggtct ggaggcggga
61 tggaacgcgg ccgccacgta caagaagaag gtggccgtcg tcgatgtgca ggcgacgcac
121 ggtccgccgt ttttcgccgc gctcggcggc acgtgcgtga acgtcggctg cgtccctaag
181 aaactcatgg tgacaggtgc ccagtacatg gacctgatcc gcgagtctgg cggcttcgga
241 tgggagatga accgcgaatc gctctgcccc aattggaaga cgctcatcgc cgcgaagaac
301 aaggtggtga acggcatcaa cgagagctac aagagcatgt ttgctgatac ggagggcctc
361 agctttcaca tgggcttcgg cgcccttcag gacgctcaca cggtgctggt gcgcaagtcg
421 gaagacccaa acagcgacgt gctggagacc ctcgacacgg agtacatcct cattgctacc
481 ggctcctggc cgacgcgcct tggagtcccc ggcgatgagt tctgcatcac gagcaacgag
541 gccttctacc tcgaagatgc ccccaagcgg atgctgtgcg tcggcggcgg ctacatcgcc
601 gtcgagtttg ccggcatctt caacggctac aagccccgcg gtggttatgt agacctgtgc
661 taccgcggcg atcttatttt gcgcggcttc gacacagagg tgcgcaagag cctgactaag
721 cagctggggg cgaacggcat acgagtgcgc accaacttga acccgacaaa aattacgaag
781 aacgaggacg gctcgaatca cgttcacttc aacgatggca cggaggagga ctacgatcag
841 gtcatgctcg cgatcggtcg cgtgccgcgc tcgcagacgc tgcagctcga caaggccggc
901 gttcaaacag caaagaacgg cgccgtgcag gtcgacgcgt actcgaagac ttcggtggac
961 aacatctacg ccatcggtga cgtgacgaac cgcgtgatgt tgacgccggt ggccatcaac
1021 gaaggcgccg ccttcgccga aaccgtcttc ggcggcaagc ctcgcgccac cgaccacacg
1081 aaggtcgcgt gcgccgtgtt ctccataccg ccgatcggca cgtgcggcat gacggaggag
1141 gaggcggcga agaaccacga aactgtcgcc gtgtacgaga gctgcttcac gccccttatg
1201 cacaacatca gcggcagcaa gcacaaggaa tttatgatcc gcatcatcac ggaccaaccc
1261 agcggcgagg tgctgggcgt tcacatgctc ggcgacagtg cgcctgagat catccagagc
1321 gtcggcattt gcatgaagat gggcgccaag atcagtgact tccacaacac catcggagtc
1381 cacccgacaa gcgccgagga gctctgctcc atgcgcactc cggcgtactt ctacgaaaat
1441 ggcaagcgcg tcgaaaagct gagcagcaac ctctga
*GC% Content for gene: 61.11%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGAGCCGTG CGTACGATCT GGTCGTTCTG GGCGCCGGAT CGGGCGGCTT GGAGGCAGGC TGGAACGCCG CTGCTACGTA TAAAAAAAAA GTCGCAGTGG TAGATGTGCA GGCGACTCAC GGCCCGCCAT TCTTTGCAGC ACTGGGAGGC ACGTGCGTGA ATGTCGGCTG CGTACCGAAA AAGCTTATGG TTACCGGAGC GCAGTATATG GATCTGATTC GCGAATCAGG GGGCTTCGGA TGGGAGATGA ATCGGGAATC CCTTTGCCCG AACTGGAAAA CTTTGATTGC TGCTAAAAAC AAAGTGGTTA ATGGCATCAA TGAATCATAT AAGTCGATGT TCGCGGATAC GGAAGGGTTA AGCTTCCATA TGGGTTTTGG CGCCCTCCAA GATGCGCACA CGGTGCTGGT ACGTAAAAGT GAAGATCCAA ATTCGGATGT CCTCGAAACC CTGGATACAG AATATATTCT TATCGCCACG GGCTCCTGGC CAACCCGCCT GGGTGTTCCG GGAGACGAAT TTTGCATCAC TAGCAACGAA GCCTTCTATT TGGAAGATGC GCCGAAACGT ATGCTTTGCG TAGGCGGTGG CTATATCGCG GTCGAATTCG CGGGCATTTT TAACGGCTAT AAGCCGCGTG GTGGTTATGT CGATCTGTGC TACCGTGGCG ATCTGATCCT GCGTGGCTTC GATACGGAAG TCCGCAAGAG TCTTACGAAA CAACTGGGAG CGAACGGTAT TCGTGTGCGC ACGAATCTCA ACCCGACCAA AATTACGAAG AACGAGGACG GTTCCAACCA TGTGCACTTC AATGATGGAA CCGAAGAGGA TTATGACCAG GTTATGCTCG CGATTGGTCG CGTTCCTCGG AGTCAGACCC TCCAACTGGA CAAGGCGGGT GTCCAAACCG CAAAAAACGG CGCGGTTCAG GTCGACGCGT ACAGCAAAAC CAGTGTCGAT AACATCTATG CTATCGGTGA CGTTACTAAC CGTGTTATGT TAACCCCGGT GGCGATCAAC GAAGGGGCCG CGTTCGCAGA AACCGTGTTC GGTGGTAAAC CCCGGGCGAC TGACCATACC AAAGTGGCTT GTGCGGTGTT CAGTATTCCG CCGATTGGTA CCTGCGGTAT GACTGAAGAG GAAGCGGCAA AAAACCATGA AACCGTTGCC GTTTACGAAA GCTGTTTCAC ACCGCTCATG CATAACATTT CGGGATCTAA GCATAAAGAA TTTATGATTC GTATCATCAC GGATCAGCCG TCAGGAGAAG TTCTGGGTGT ACACATGCTG GGGGATAGTG CCCCAGAAAT TATTCAGTCT GTTGGTATTT GTATGAAAAT GGGAGCCAAA ATTAGCGATT TTCACAATAC GATTGGGGTG CATCCGACGA GCGCAGAAGA GCTTTGTTCG ATGCGCACGC CGGCTTATTT TTACGAGAAC GGTAAGCGTG TGGAAAAACT GAGCTCAAAT CTTTAA
*GC% Content for gene (codon optimized): 51.08%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
"Leishmania Major » Protozoan and Helminth Parasites » Pathogen Profile Dictionary."Leishmania Major » Protozoan and Helminth Parasites » Pathogen Profile Dictionary. . <http://www.ppdictionary.com/parasites/major.htm>. (Accessed 4/25/2013)
See ProtocolTargetDiscoveryVDS.docx for more
Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf